Transcriptional regulation in cells is a complex biological process, regulated by multiprotein complexes including transcription factors, coactivators and repressors, receptors as well as platform/DNA binding proteins. One basic principle in transcriptional regulation is based on the posttranslational modification of histone proteins, namely histone proteins H2A/B, H3 and H4 forming the octameric histone core complex. The complex N-terminal modifications at lysine residues by acetylation or methylation and at serine residues by phosphorylation constitute part of the so called “histone code” (Strahl & Ellis, Nature 403, 41-45, 2000). In a simple model, acetylation of positively charged lysine residues decreases affinity to negatively charged DNA, which now becomes accessible for the entry of transcription factors.
Histone acetylation and deacetylation is catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). In many cases, HDACs are associated with transcriptional repressor complexes, switching chromatin to a transcriptionally inactive, silent structure (Marks et al. Nature Cancer Rev 1, 194-202, 2001). The opposite holds true for HATs, which are frequently associated with transcriptional activator complexes. Nevertheless, opposite functions for HATs and HDACs have been described in the literature. The cAMP response element binding protein (CBP) and p300 as HATs contain the transcriptional repressor domain CRD1 (cell cycle regulatory domain 1), allowing these proteins to act as transcriptional repressors (Snowden et al., Mol Cell Biol 20, 2676-2686, 2000). Transcriptional signatures of HDAC inhibitors show a similar proportion of induced and repressed genes. In one study, HDAC inhibition abrogates interferon-induced gene transcription presumably by antagonizing the co-activator function of HDAC1 for the interferon stimulated gene factor 3 (ISGF3; Nusinzon & Horvath, Science STKE August 2005). In melanoma cells, interaction of NfκB p65 with STAT1 is dependent on STAT1 acetylation. HDAC inhibitor or interferon α mediated STAT1 hyperacetylation causes the cytoplasmatic retention of NfκB, finally leading to repression of NFκB regulated genes (Krämer et al. Gen Develop 20, 473-485, 2006).
Three different classes of HDACs have been described so far, namely class I (HDAC 1-3, 8) with Mr=42-55 kDa primarily located in the nucleus and sensitive towards inhibition by Trichostatin A (TSA), class II (HDAC 4-7, 9, 10) with Mr=120-130 kDa and TSA sensitivity and class DI (Sir2 homologues) which are quite distinct by their NAD+ dependency and TSA insensitivity (Ruijter et al. Biochem. J. 370, 737-749, 2003; Khochbin et al. Curr Opin Gen Dev 11, 162-166, 2001; Verdin et al. Trends Gen 19, 286-293, 2003). HDAC 11 with Mr=39 kDa displayed homology to class I and II family members (Gao et al. J Biol Chem 277,25748-25755, 2002) and is now defined as the sofar only class IV member (Gregoretti et al. J Mol Biol 338, 17-31, 2004). HATs and HDACs exist in large complexes together with transcription factor and platform proteins in cells (Fischle et al. Mol Cell 9, 45-47, 2002).
Substrates different to histone proteins exist. For HDACs these include transcription factors like p53 STAT proteins and TFII E, α-tubulin as a major protein of microtubles, or chaperones like heat shock protein 90 (Hsp90; Johnstone & Licht, Cancer Cell 4, 13-18, 2003). Therefore the correct name for HDACs would be lysine-specific protein deacetylases. As a consequence of these findings, inhibitors of HDACs affect not only chromatin structure and gene transcription but also protein function and stability by regulating protein acetylation in general. This function of HDACs in protein acetylation might also be important for understanding of immediate gene repression by treatment with HDIs (von Lint et al. Gene Expression 5, 245-253, 1996). In this regard, proteins involved in oncogenic transformation, apoptosis regulation and malignant cell growth are of particular importance.
Different publications highlight the pathophysiological importance of reversible histone acetylation for cancer drug development (reviewed by Kramer et al. Trends Endocrin Metabol 12, 294-300, 2001; Marks et al. Nature Cancer Rev 1, 194-202, 2001; Minucci & Pelicci, Nature Rev Cane 6, 38-51, 2006; Yoo, & Jones Nat. Rev. Drug Discov. 5, 37-50 2006):                (i) Mutations of CBP as a HAT are associated with Rubinstein-Taybi syndrome, a cancer predisposition (Murata et al. Hum Mol Genet 10, 1071-1076, 2001),        (ii) Aberrant recruitment of HDAC1 activity by transcription factors in acute promyelocytic leukemia (APL) is mediated by the PML-retinoic acid receptor α fusion gene (He et al. Nat genet 18, 126-135, 1998)        (iii) Aberrant recruitment of HDAC activity by the overexpressed BCL6 protein was shown in non-Hodgkins lymphoma (Dhordain et al. Nucleic Acid Res 26, 4645-4651, 1998) and finally        (iv) Aberrant recruitment of HDAC activity by the AML-ETO fusion protein was shown for acute myelogenous leukemia (AML M2 subtype; Wang et al. Proc Natl Acad Sci USA 95, 10860-10865, 1998). In this AML subtype, the recruitment of HDAC1 activity causally leads to gene silencing, a differentiation block and oncogenic transformation.        (v) HDAC1 gene knock-out in mice showed that HDAC1 has a profound function in embryonal stem cell proliferation by repressing cyclin-dependent kinase inhibitors p21waf1 and p27kip1 (Lagger et al. Embo J. 21, 2672-2681, 2002). Since p21waf1 is induced by HDIs in many cancer cell lines, HDAC1 might be a crucial component in cancer cell proliferation as well. Initial siRNA based gene-knock down experiments in HeLa cells support this hypothesis (Glaser et al. 310, 529-536, 2003)        (vi) HDAC2 is overexpressed in colon carcinoma upon constitutive activation of the wnt/β-catenin/TCF signalling pathay by loss of functional adenomatosis polyposis coli (APC) protein as reported by Zhu et al. (Cancer Cell 5, 455-463, 2004)        (vii) A high expression of HDAC1, 2 and 3 in prostate adenocarcinomas was shown by immunohistochemistry, with HDAC2 as an independent prognostic factor for patient survival and HDAC1/2 correlating positively with tumor grade (Roeske et al, EORTC-NCI-AACR meeting Prague 2006, Abstract 350)        
About 2%-3% of all genes are regulated by histone acetylation as estimated by differential display analysis and array based whole genome studies (von Lint et al. Gene Expression 5, 245-253, 1996, Sasakawa et al. Biochem Pharmacol 69, 603-16, 2005). Studies with the HDAC classI/II selective inhibitor suberoylanilide hydroxamic acid (SAHA) in multiple myeloma cells showed that these transcriptional changes can be grouped into distinct functional gene classes important for e.g. regulation of apoptosis or proliferation (Mitsiades et al. Proc Natl Acad Sci 101, pp 540, 2004). In a different study with the natural compound KF228 (Depsipeptide) and the cancer cell lines ACHN (renal cancer), PC3 (prostate cancer) and U937 (histiocytic leukemia), 105 up- and 100 down-regulated genes were identified (Sasakawa et al. Biochem Pharmacol 69, 603-16, 2005). Genes encoding proteins important for cell cycle/mitosis regulation (e.g. p21waf1, CyclinA2, SAK, MKLP1), chromatin structure (e.g. histone proteins like histone H1, apoptosis/survival (e.g. caspase 9, TNF/TNFR family members), protein turnover (e.g. ubiquitin ligase E2H) or mitogenic/stress signaling (e.g. MKK3, MAPKAPK3, Ki67) were induced or repressed by treatment with FK228. Two of these genes (caspase 9 and mitogen activated protein kinase phosphatase 1/MKP1 were used as biomarkers to predict the response of human tumors xenografted onto nude mice to therapy with FK228 (Sasakawa et al. Biochem Pharmacol 69, 603-16, 2005). In a different study, the transcriptional changes in the acute human T-lymphoblastic leukemia cell line CCRF-CEM by SAHA and FK228 were studied (Peart et al. Proc Natl Acad Sci 102, 3697-3702, 2005). According to the experimental and statistical condition applied, the expression of 22.1% (SAHA) and 24.8% (FK228) of analyzed genes (about 10.000 unique accession numbers) were changed. Only a small subset of genes was identified that discriminated between both HDAC inhibitors. Finally, HDI regulated genes were functionally clustered showing alterations genes encoding protein involved, for example, in apoptosis regulation (e.g. caspases 3 and 5, APAF1, BCL XL, IKB, TNF) and proliferation (e.g. cyclins G1, G2, E1, B2, E2 and CKD2, CDC25c).
HDAC inhibitors arrest cells at G1 and G2/M within the cell cycle and deplete S-phase cells, as shown for Depsipeptide as an example (Sandor et al., British J Cancer 83, 817-825, 2000). The interaction of HDAC3 with the mitotic kinase Aurora B was shown recently (Li et al. Genes Dev. 20:2566-79, 2006). In this study, phosphorylation of histone H3 at S10 by Aurora B was dependent on HDAC3 mediated N-terminal histone H3 deacetylation, giving a hint to the partial M-phase arrest seen by many HDIs.
HDAC inhibitory compounds induce p53 and caspase3/8 independent apoptosis and have broad anti-tumor activity. Anti-angiogenic activity was described also, which might be related to down-regulation of VEGF and HIF1α. In summary, HDAC inhibition affects tumor cells at different molecular levels and multiple cellular proteins are targeted.
Interestingly, HDAC inhibitors were found to induce cellular differentiation and this pharmacological activity might contribute to their anti-cancer activity as well. For example it was shown recently that suberoylanilide hydroxamic acid (SAHA) induces differentiation of breast cancer cell lines, exemplified by resynthesis of milk fat membrane globule protein (MFMG), milk fat globule protein and lipid (Munster et al. Cancer Res. 61, 8492, 2001). Also, induction of fetal hemoglobin synthesis in hematopoietic cells of the erythrocyte lineage has been studied in a clinical trial with the butyrate analog AN-9 (Patnaik et al. Clin Cancer Res. 8(7): 2142-8, 2002).
There is growing rational for synergism of HDAC inhibitors with chemotherapeutic as well as target specific cancer drugs. For example, synergism was shown for (i) SAHA with the kinase/cdk inhibitor flavopiridol (Alemenara et al. Leukemia 16, 1331-1343, 2002) or with the death receptor DR4/5 ligand TRAIL (Butler et al. Int J Cancer 119, 944-54, 2006; Sonnemann et al. Invest New drugs 23, 90-109, 2005), (ii) for LAQ-824 with the bcr-abl kinase inhibitor Glivec in CML cells (Nimmanapalli et al. Cancer Res. 63, 5126-5135, 2003) or the KDR/VEGFR2 kinase inhibitor PTK787/ZK222584 in angiogenesis (Qian et al. Cancer Res. 64, 6626-34, 2004), (iii) for SAHA and Trichostatin A (TSA) with etoposide (VP16), cisplatin and doxorubicin (Kim et al. Cancer Res. 63, 7291-7300, 2003), for TSA in combination with retinoid acid in acute myeloid leukemia/AML (Ferrara et al. Canc Res 61, 2-7, 2001) (iv) for LBH589 with the Hsp90 inhibitor 17-allyl-amino-demethoxy-geldanamycin (17-AAG; George et al. Blood online, Oct. 28, 2004) or the proteasome inhibitor bortezomib/Velcade (Maiso et al. Canc Res 66, 5781-5789, 2006, (iiv) PXD101 with 5-FU in colon cancer models (Tumber et al. Canc Chem Pharmacol nov. 2006) and Taxol or Carboplatin in ovarian carcinoma models (Qian et al. Mol Canc ther 5, 2086-95) Also it was shown that HDAC inhibition causes reexpression of estrogen or androgen receptors in breast and prostate cancer cells with the potential to resensitize these tumors to anti-hormone therapy (Yang et al. Cancer Res. 60, 6890-6894, 2000; Nakayama et al. Lab Invest 80, 1789-1796, 2000). Finally, histone deacetylase inhibitors sensitize towards cellular radiation responses as reviewed recently (Karagiannis & El-Osta, Oncogene 25, 3885-93, 2006).
HDAC inhibitors from various chemical classes were described in the literature with four most important classes, namely (i) hydroxamic acid analogs, (ii) benzamide analogs, (iii) cyclic peptides/peptolides and (iv) fatty acid analogs. A comprehensive summary of known HDAC inhibitors was published by various authors (Miller et al. J Med Chem 46, 5097-5116, 2003; Dokmanovic & Marks, J Cell Biochem 96, 293-304, 2005; Drummond et al. Ann Rev Pharmacol Toxicol 45, 495-528, 2005; Bolden J E, et al. Nat Rev Drug Discov 5:769-784, 2006; Sorbera, Drugs of the Future 31, 335-344, 2006). There is only limited data published regarding specificity of these histone deacetylase inhibitors. In general most hydroxamate based HDI are not specific regarding class I and II HDAC enzymes. For example TSA inhibits HDACs 1, 3, 4, 6 and 10 with IC50 values around 20 nM, whereas HDAC8 was inhibited with IC50=0.49 (Tatamiya et al, AACR Annual Meeting 2004, Abstract #2451). But there are exceptions like the experimental HDI Tubacin, selective for the class II enzyme HDAC 6 (Haggarty et al. Proc. Natl. Acad. Sci. USA 100, 4389-4394, 2003). In addition, data on class I selectivity of benzamide HDIs are emerging. MS-275 inhibited class I HDAC1 and 3 with IC50=0.51 μM and 1.7 μM, respectively. In contrast class II HDACs 4, 6, 8 and 10 were inhibited with IC50 values of >100 μM, >100 μM, 82.5 mM and 94.7 μM, respectively (Tatamiya et al, AACR Annual Meeting 2004, Abstract #2451). Comparable data were published by Hu et al. with inhibition of IC50=0.3 μM, 8 μM and >100 μM for HDAC1, 3 and 8, respectively (Hu et al. J Pharmacol Exp Therap 307, 720-28, 2003). Finally, the benzamide analog MGCD0103 inhibited HDAC1, 2, 3 and 11 with IC50 from 0.1-2 μM and HDACs 4 to 8 with IC50 values >20 μM (Kalita et al. AACR-NCI-EORTC Conference Philadelphia 2005; Abstract C216).
Clinical studies in cancer with HDAC inhibitors are on-going, namely with SAHA (Zolinza™ by Merck Inc.; Kelly et al. J Clin Oncol 23, 3923-31, 2005), CRA-024781 (Pharmacyclics Inc.; Buggy et al, Mol Canc Therap 5, 1309-17, 2006), ITF-2357 (Italfarmaco; J Hepatology 42, 210-17, 2005), Valproic acid (Topotarget; Göttlicher et al. EMBO J. 20, 6969-78, 2001), FK228/Depsipeptide (Gloucester Pharmaceuticals/NC; Nakajima et al. Exp Cell Res 241, 126-33, 19981), MS275 (Berlex-Schering; Ryan et al. J Clin Oncol 23, 3912-22, 2005), NVP LBH-589 (Novartis; Remiszewski et al. J Med Chem 46, 4609-24, 2003), PXD-101 (Topotarget/Curagen; Plumb et al. Mol Canc Therap 2, 721-28, 2003), and MGCD0103 (Methylgene Inc.; Kalita et al. AACR-EORTC-NCI meeting 2005, Abstract C216). These studies showed evidence of clinical efficacy, highlighted recently by partial and complete responses with FK228/Depsipeptide in patients with peripheral T-cell lymphoma (Plekarz et al. Blood, 98, 2865-2868, 2001) and approval in this indication of SAHA (Zolinza™) by Merck & Co., Inc. (Nature Biotechn 25, 17-18, 2007).
Recent publications also showed possible medical use of HDAC inhibitors in diseases different to cancer. These diseases include systemic lupus erythematosus (Mishra et al. J Clin Invest 111, 539-552, 2003; Reilly et al. J. Immunol. 173, 4171-4178, 2004), rheumatoid arthritis (Chung et al. Mol Therapy 8, 707-717, 2003; Nishida et al. Arthritis & Rheumatology 50, 3365-3376, 2004), inflammatory diseases (Leoni et al. Proc Natl Acad Sci USA 99, 2995-3000, 2002), neurodegenerative diseases like Huntington's disease (Steffan et al. Nature 413, 739-743, 2001, Hockly et al. Proc Natl Acad Sci USA 100(4): 2041-6, 2003), cardiac hyperthrophy (Kong et al., Circulation 113, 2579-88, 2006), muscle dystrophy (Minetti et al. Nat Med 12, 1147-50, 2006), adipositas (Lagace & Nachtigal, J Biol. Chem. 279, 18851-860, 2004) and diabetes (Gray & DeMeyts, Diabetes Metab Res Rev 21, 416-33, 2005).
It is known that the loss of histone acetyltransferase (HAT) activity or increased histone deacetylase (HDAC) function leads to neuronal dysfunction and degeneration. Consistent with this fact, HDAC inhibition can restore the HAT-HDAC balance in the CNS in favour of HAT activity, which may facilitate survival, reduce inflammation, and neuronal damage. HDAC inhibitors such as SAHA, TSA, and sodium butyrate have been shown to promote neuronal survival. Thus, HDAC inhibitors have potential applications for the treatment or as adjuncts in neurodegenerative disorders such as stroke, Huntington's disease, Alzheimer's disease, and other such CNS disorders (Langley, B.; Gensert, J. M.; Beal, M. F.; Ratan, R. R. Current Drug Targets—CNS & Neurological Disorders 2005, 4, 41-50).
In fungi such as Candida albicans, HDAC enzymes HDA1 and RPD3 are known to contribute to its virulent character. Some HDAC inhibitors such as SAHA have been shown to inhibit the fluconazole induced resistance induction in Candida cultures (Mai, A.; Rotili, D.; Massa, S.; Brosch, G.; Simonetti, G.; Passariello, C.; Palamara, A. T. Bioorg. Med. Chem. Lett. 2007, 17, 1221-1225). The biological activity of HDAC inhibitors also comprises antiprotozoal, antifungal, and antiviral effects. The HDAC inhibitor TSA initially was discovered as an antifungal antibiotic. Apicidin, a different natural compound type of a HDAC inhibitor, and its derivatives have been tested and shown to be effective antimalarial agents and according to one study, an increase in their HDAC inhibitory activity correlated with their improved antimalarial activity (Meinke, P. T.; Liberator, P. Current Medicinal Chemistry 2001, 8, 211-235).
Cancer chemotherapy was established based on the concept that cancer cells with uncontrolled proliferation and a high proportion of cells in mitosis are killed preferentially. Standard cancer chemotherapeutic drugs finally kill cancer cells upon induction of programmed cell death (“apoptosis”) by targeting basic cellular processes and molecules, namely RNA/DNA (alkylating and carbamoylating agents, platin analogs and topoisomerase inhibitors), metabolism (drugs of this class are named anti-metabolites) as well as the mitotic spindle apparatus (stabilizing and destabilizing tubulin inhibitors). Inhibitors of histone deacetylases (HDIs) constitute a new class of anti cancer drugs with differentiation and apoptosis inducing activity. By targeting histone deacetylases, HDIs effect histone (protein) acetylation and chromatin structure, inducing a complex transcriptional reprogramming, examplified by reactivation of tumor suppressor genes and repression of oncogenes. Beside affecting acetylation of N-terminal lysine residues in core histone proteins, also non-histone targets important for cancer cell biology are modified by acetylation at lysine residues. These non-histone substrates are, for example, heat-shock-protein 90 (Hsp90; Bali et al. J Biol Chem 280, 26729-734, 2005), α-tubulin (Hubbert et al. Nature 417, 455-58, 2002), STAT1 or STAT3 (Yuan et al. Science 307, 269-273, 2005; Krämer et al. Gen & Develop 20, 473-485, 2006) or the p53 tumor suppressor protein (Mol Cell 24, 807-808, 2006). The medical use of HDIs might not be restricted to cancer therapy, since efficacy in animal models for eg, rheumatoid arthritis, neurodegeneration, cardiac hyperthrophy and muscle dystrophy was shown.